Cancer is a disease characterized in that cells grow disorderly. A major factor in determining the fatality rate of cancer is growth in a metastatic lesion rather than growth in a primary lesion where the cancer has initially developed. The actual 5-year survival rate of patients diagnosed with metastatic cancer is reportedly 20% or less. The prevention of cancer metastasis is a clinically great challenge.
It has been clinically accepted since a long time ago that cancer cell-dependent platelet aggregation is involved in the hematogenous metastasis of cancer. The group of the present inventor have established a highly metastatic cancer cell line exhibiting platelet aggregation-inducting activity, and identified a platelet aggregation-inducing factor, Aggrus, expressed on the cell membrane surface of the highly metastatic cancer cell line (Patent Literature 1 and Non Patent Literature 1).
The platelet aggregation-promoting factor Aggrus (also known as podoplanin, gp44, T1α) is a type-I transmembrane protein and is known to be overexpressed in various cancers including squamous cell carcinoma, mesothelioma, Kaposi sarcoma, testicular tumor, brain tumor, and bladder cancer (Non Patent Literatures 2 to 13).
Recently, C-type lectin-like receptor (CLEC-2) expressed on platelet has been identified as one of the counterpart receptors of Aggrus (Non Patent Literature 14). It is known that the binding of CLEC-2 to Aggrus expressed on tumor cells activates platelet-aggregation signals in the platelet even in the absence of plasma components to induce platelet aggregation. In other words, a substance inhibiting the binding between Aggrus and CLEC-2 is considered to inhibit platelet aggregation and inhibit cancer metastasis. Thus, the substance inhibiting the binding between Aggrus and CLEC-2 is expected to be applied to the treatment of cancer or thrombosis. In actuality, the present inventors disclose that monoclonal antibodies or low-molecular compounds against Aggrus inhibiting the interaction between Aggrus and CLEC-2 can inhibit platelet aggregation or cancer metastasis (Patent Literatures 2 and 3 and Non Patent Literature 15).
Monoclonal antibodies can specifically bind to cell surface antigens and can thereby cause immunological response to the target cells. By use of this reaction, many monoclonal antibodies are used in the treatment of cancer or the treatment of immunological disease. The monoclonal antibodies exhibit a therapeutic effect via three typical modes of action: neutralization, antibody-dependent cellular cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC).
The present inventors have previously revealed that all anti-Aggrus monoclonal antibodies P2-0 (Accession No. FERM BP-11446 deposited on Feb. 18, 2011), MS-1 (Accession No. FERM BP-11447 deposited on Dec. 28, 2011), MS-3 (Accession No. FERM BP-11448 deposited on Dec. 28, 2011), and MS-4 (Accession No. FERM BP-11449 deposited on Dec. 28, 2011) deposited with International Patent Organism Depositary National Institute of advanced Industrial Science and Technology, AIST Tsukuba Central 6, 1-1, Higashi 1-chome Tsukuba-shi, Ibaraki-ken, 305-8566, Japan, produced by established hybridomas are neutralizing antibodies that inhibit Aggrus-CLEC-2 binding, and exhibit effects of inhibiting platelet aggregation, inhibiting cancer metastasis, and inhibiting tumor growth in an ADCC activity-independent manner (Patent Literature 2).
Among these four antibodies, both the P2-0 and MS-1 antibodies having high activity of inhibiting Aggrus-CLEC-2 binding are antibodies recognizing intraregional amino acids of a PLAG domain (platelet-aggregation stimulating domain) as an epitope. The PLAG domain is an amino acid sequence having a consensus sequence EDXXVTPG (SEQ ID NO: 15) conserved across species, including humans, mice, and rats. The PLAG domain is an amino acid sequence containing three tandem repeats of the PLAG domain (PLAG1 to PLAG3) in proximal regions, and, in the case of human Aggrus, is located in a region from positions 29 to 54 of the amino acid sequence.
The PLAG domain is known to be present extracellularly and involved in platelet-aggregating activity. Results of co-crystal structure analysis on Aggrus and CLEC-2 show that glutamic acid at position 47 and aspartic acid at position 48 as well as sialic acid attached to threonine at position 52 in Aggrus binds to four arginine residues present in a region from positions 107 to 157 of CLEC-2 (Non Patent Literature 16). The P2-0 and MS-1 antibodies developed by the present inventors also recognize, as an epitope, a region from positions 45 to 49 of the amino acid sequence of Aggrus, which overlaps with the CLEC-2-binding region of Aggrus. The MS-3 and MS-4 antibodies recognize, as an epitope, a region from positions 54 to 61 of the amino acid sequence of Aggrus, which is located in proximity to the CLEC-2-binding region of Aggrus.